We have continued to develop AAV4 and AAV5 as vectors for gene transfer and they are both actively being evaluated in several gene therapy applications including gene transfer to the lung, CNS, eye, and salivary gland. In addition to distributing these vectors to labs throughout the world, we have continued to collaborate with a number of researchers in the field and in several publications describe the use of AAV4 and AAV5 based vectors to express beta glucaronidase for the correction of learning defects in MPS VII mice (Liu et al. 2007) (Liu et al. 2005), transfer BMPs to muscle to induce bone repair (Li et al 2006), transduce primary salivary epithelial cells to repair damage to the gland or produce therapeutic proteins (Tran et al. 2007, Voutetakis et al. 2007), and deliver a truncated CFTR for primary airway epithila from cystic fibrosis patients and correct the underlying defect in chloride transport associated with this disease (Ostegaard et al 2005). Vectors based on BAAV, which we have previously cloned and characterized, also demonstrating utility in defining the underlying biology associated with signal transduction and channel formation in the inner ear (Ortolano et al. 2008) and may also be useful in treating hereditary deafness and balance disorders. We also are working with both intramural and extramural researches to test the effect of local expression of immunomodulatory proteins on animal models of Sjogrens Syndrome to evaluate them as potential therapies for this disease. In addition through funding received from the office of the scientific director as part of the salivary gland biology initiative, we have begun to collaborate with researchers in the Laboratory of Cell and Developmental Biology, NIDCR at identifying AAV vectors for gene transfer to the developing salivary gland. [unreadable] We believe that just as our characterizations of AAV4, AAV5, and BAAV have advanced the field of gene therapy, the development of new vectors also will have an impact on other gene therapy applications, as well as our understanding of parvovirus biology. As a first step in this endeavor, we have developed a PCR based method for rapidly identifying and cloning divergent AAV isolates. While we have found a number of new isolates we have focused our efforts on those stocks that i) may have originated from epithelial cells which is in line with our goal of improving gene transfer to secretory epithilia such as the salivary glands, and ii) have minimal immunological reactivity with pooled human serum. The first isolate we have extensively characterized that meets these criteria is an isolate found as a contaminant in a Simian Adenovirus 17 (SV17) sample obtained from vervet monkeys. We have termed this isolate AAV12. In contrast to all other reported AAVs, AAV12 cell attachment and transduction does not require cell surface sialic acids or heparan sulfate proteoglycans (HSPG). Furthermore, rAAV12 is resistant to neutralization by circulating antibodies from human serum. Feasibility of rAAV12 as a vector was demonstrated in a mouse model in which muscle and salivary glands were transduced. These characteristics make rAAV12 an interesting candidate for gene transfer applications (Schmidt et al. 2008). In addition to providing new vectors for gene transfer, study of these new isolates has provided important information regarding structural domains on the surface of the virus[unreadable] Our knowledge of the interactions necessary for cellular transduction with AAV4 and AAV5 clearly has been helpful in defining their biology and likely will identify novel applications for these vectors. Furthermore, the tools we have developed while examining AAV4 and AAV5 tropism can be applied to other novel vectors we have isolated. We expect this information will aid in identifying optimal target cells for the different AAV vectors and also aid in the development of a new generation of vectors for gene transfer with very defined cell tropism. [unreadable] While natural isolates have served as a rich source of vectors for gene transfer, an alternative approach would be to develop vectors with defined tropism. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. Previously we have reported the structure of the AAV5 capsid and more recently we have solved the structure for one of the most antigenically distinct serotypes, AAV4, to 3- resolution.[unreadable] Structural comparison of AAV4 to AAV2 shows conservation of core beta strands and helical secondary structure elements, which also exist in all other known parvovirus structures. However, surface loop variations, some containing compensating structural insertions and deletions in adjacent regions, result in local topological differences on the capsid surface. The observed differences in loop topologies at subunit interfaces are consistent with the inability of AAV2 and AAV4 VPs to combine for mosaic capsid formation in efforts to engineer novel tropisms. Significantly, all of the surface loop variations are associated with amino acids reported to affect receptor recognition, transduction, and anticapsid antibody reactivity for AAV2. This observation suggests that these capsid regions may also play similar roles in the other AAV serotypes. [unreadable] To date over 100 AAV capsid sequences have been cloned from a variety of sources but we have very little understanding of their host cell requirements for entry and even less of an understanding of which regions on the surface of the particles are involved in entry. We recently reported the identification of a new heparin dependent transduction domain on the surface of AAV(VR-942) that is distinct from the heparin binding site in AAV2 (Schmidt 2008). This is only the second domain on the surface of any AAV particle for which a receptor function has been mapped. Mapping the heparin binding domain on AAV2 has allowed for this region to be deleted and the tropism of these particles to be retargeted. This finding has had a major impact on the field and now knowledge routinely used by several labs. Further analysis of the AAV(VR-942) heparin transduction region is likely to also lead to improvements in gene transfer vectors. [unreadable] Sjogrens syndrome is an autoimmune disease, characterized by lymphoid cell infiltration into the salivary and lacrimal glands, and affects 0.5% of the population in the United States of which 90% are women. The consequence of chronic immune cell activation in these exocrine glands is diminished secretory function, which leads to symptoms of dry mouth and dry eyes. We have demonstrated that delivery of immunomodulatory proteins to the salivary glands of a mouse model of Sjogrens Syndrome can amelorate the gland dysfunction in these animals. In the past we have used female non-obese diabetic (NOD) mouse to model the human disease. While these mice develop exocrine gland infiltrates and, as in SS, a loss of salivary flow that is age and gender dependent, they also develop insulin dependent type 1 diabetes which complicates the study of Sjogrens Syndrome. Furthermore, maintenance of longitudinal stability with respect to onset of the Sjogrens Syndrome phenotype has been reported as problematic. Currently other mouse models of this disease are available and we are reviewing and evaluating them as an alternative to the NOD mouse. [unreadable] In summary, the future directions for the AAV Biology Section will be to continue examination and development of gene transfer vectors for use in treating disease as well as refine our tools for studying interactions necessary for cellular transduction. We will continue to identify the domains critical for these interaction on the virus surface, as well as to identify new AAV isolates that maybe useful for gene therapy applications.